A variety of techniques are currently available for efficient amplification of nucleic acids even from a few molecules of a starting nucleic acid template. These include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), and rolling circle amplification (RCA). Many of these techniques involve an exponential amplification of the starting nucleic acid template and are able to generate a large number of amplified products quickly. Kits for the amplification of a target nucleic acid are commercially available (e.g., GenomiPhi™ (General Electric, Inc.) and RepliG™ (Qiagen, Inc.), but improvements to these methods would be advantageous.
Nucleic acid amplification techniques are often employed in nucleic acid-based assays used for analyte detection, sensing, forensic and diagnostic applications, genome sequencing, whole-genome amplification, and the like. Such applications often require amplification techniques having high specificity, sensitivity, accuracy, and robustness. Most of the currently available techniques for nucleic acid amplification, however, suffer from high background signals, which are generated by non-specific amplification reactions yielding unwanted amplification products. These non-specific amplification reactions hinder effective utilization of many of these techniques in critical nucleic acid-based assays. For example, use of a traditional amplification reaction may produce a false-positive result, thereby leading to an incorrect diagnosis. Such non-specific, background amplification reactions become even more problematic when only trace amounts of the target nucleic acid to be amplified are available (e.g., whole-genome amplification from a single DNA molecule).
Non-specific, background amplification reactions may be due to, for example, amplification of a contaminating nucleic acid sequence in the sample, primer-dimer formation, or production of chimeric nucleic acids (e.g., resulting from self-hybridization of the desired nucleic acid products). A frequent source of non-specific amplification in a nucleic acid amplification reaction results from various undesirable primer interactions. A primer may hybridize to regions of a nucleic acid in either a target nucleic acid or in a contaminating nucleic acid that share some homology with a portion of the target nucleic acid. If the 3′ end of a primer has sufficient homology to an untargeted region, this region may be amplified.
Non-specific amplification may also result from unintended nucleic acid template-independent primer-primer interactions. Primers may form primer-dimer structures by intra- or inter-strand primer annealing (e.g., intra-molecular or inter-molecular hybridizations), resulting in amplification of unwanted nucleic acids. The resultant spurious primer extension products may be further amplified and may at times predominate, inhibit, or mask the desired amplification of the targeted sequence. Moreover, the amplification products may self-hybridize, allowing the nucleic acid polymerase to generate hybrid products or chimeric products during the amplification reaction.
For priming DNA synthesis, current MDA formulations often utilize random hexamers with the sequence 5′-NNNNN*N, where “N” represents a deoxyadeno sine (dA), deoxycytidine (dC), deoxyguano sine (dG), or deoxythymidine (dT) and “*” represents a phosphorothioate linkage.
Constrained-randomized hexamer primers that cannot cross-hybridize via intra- or inter-molecular hybridization (e.g., R6, where R=A/G) have been used for suppressing primer-dimer structure formation during nucleic acid amplification. These constrained-randomized primers, however, impart considerable bias in nucleic acid amplification reaction. Such primers are also of limited use for sequence-non-specific or sequence-non-biased nucleic acid amplification reactions (e.g., whole genome or unknown nucleic acid sequence amplification).
For priming DNA synthesis, MDA formulations frequently utilize random hexamers with the sequence 5′-NNNNN*N, where “N” represents a deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG), or deoxythymidine (dT) and “*” represents a phosphorothioate linkage. One solution to minimizing competing non-target nucleic acid (i.e., template DNA) amplification is to modify the oligonucleotide primers in such a way as to inhibit their ability to anneal with one another.
Previous research to overcome the issues associated with nucleic acid amplification using random hexamer primers described above includes those methods and kits disclosed in U.S. Pat. No. 7,993,839 (issued Aug. 9, 2011). The techniques described in this patent include but are not limited to the use of primers that are hexamers of the general structure 5′-+W+WNNN*S-3′, where “+” precedes a locked nucleic acid base (i.e., “an LNA base”; for example, +A=an adenosine LNA molecule), “W” represents a mixture of only dA and dT, and “S” represents a mixture of only dC and dG. The “*” represents a phosphorothioate linkage between the two nucleotides. Since “W” bases are unable to stably pair with “S” bases, the formation of the oligonucleotide duplex is inhibited, which leads to decreased amplification of non-template nucleic acids. These methods and kits may be referred to as “SD GenomiPhi.”
One improvement to the speed and sensitivity of MDA when amplifying trace nucleic acid samples is the incorporation of LNAs into the oligonucleotide primers. LNAs are a class of conformationally restricted nucleotide analogues that serve to increase the speed, efficiency, and stability of base pairing, thereby promoting the hybridization of the modified oligonucleotides to their target sequences in the nucleic acid of interest. For each LNA monomer incorporated into an oligonucleotide primer, the duplex melting temperature (Tm) is increased by 2-8° C. The increase in Tm of the duplex allows the MDA reaction to be performed under more stringent conditions, such as at a higher temperature or with a lower concentration of salt (e.g., 15 mM KCl as opposed to the 75 mM KCl used in traditional amplification reactions with unmodified primers). While the kinetics of amplification using MDA are dramatically increased by incorporation of LNAs into the random primers, one drawback is that the hexamers also anneal to each other more efficiently, leading to amplification of unwanted nucleic acids (e.g., primer-dimers).
The problems associated with the undesirable amplification of non-target nucleic acids have also been approached from the standpoint of removing contaminating nucleic acids from reagents and reagent solutions used in nucleic acid amplification methods. Kits and methods for generating nucleic acid contaminant-free reagents and reagent solutions for use in nucleic acid amplification are disclosed in U.S. Patent Application Publication No. 2009/0155859. Such methods include processing of polymerase solutions, nucleotide solutions, and primer solutions to render contaminating nucleic acids inert. The methods employ the proofreading activity of the polymerase and/or exonucleases to decontaminate the reagents and reagent solutions. The methods described in U.S. Patent Application Publication No. 2009/0155859 may at times be referred to as “Clean GenomiPhi” or “Clean GPhi.”
Despite these advancements, there remains a need for developing more efficient nucleic acid amplification methods that have lower bias in terms of sequence coverage and produce lower levels of non-specific, background amplification. Development of primers for nucleic acid amplification without sequence bias that also reduce primer-primer interaction and minimize the production of chimeric nucleic acids (e.g., unwanted nucleic acid products resulting from the annealing of the hexamer primers to the target nucleic acid amplification products) is needed in the art.